Fremont “forager” research was stimulated by the work of Steve Simms at Topaz Slough m Utah’s west desert. Topaz Slough was characterized as a temporary Fremont occupation and seemed evidence of an adaptation quite different from the more often investigated Fremont village sites. As a consequence of Shnms’ findings, which but on the work of Madsen , the term ‘Fremont foragers’ has become something of a buzz phrase in Fremont studies Despite the clear interest in Fremont strategic variability and the fact that many west desert open sites with Fremont occupations are known , few have been excavated, and reports of that work have tended to be brief . The present monograph, reporting the excavation of a “Fremont village” on a large dune on the extreme southwest margm of the Great Salt Lake Desert, is the first detailed site report of an extensively excavated Fremont forager site. This review briefly describes the contents of the monograph by chapter and comments on the contributions of the work. The Introduction states the reasons for the Buzz-Cut Dune excavations. The site was discovered during an effort to blade the top off a large dune to improve the one-of-sight between communication towers at Dugway Proving Ground. This activity exposed several possible structures, some of which contained Fremont diagnostics.
In order to salvage the clearly significant site information. Anthrax is a severe infectious disease caused by Bacillus anthracis. The spores can be produced easily and released in air as a biological weapon,plastic potting pots leading to a fatality rate of 86–89% . Bacillus anthracis secrets anthrax toxin, which is composed of a cell-binding protein, namely protective antigen , and two enzymatic proteins called lethal factor and edema factor . The cellular toxicity starts with the binding of PA to anthrax toxin receptors, after which the bound PA is cleaved by a furin family protease, leaving a 63 kDa fragment bound to the receptors . The receptor-PA complex then self-assembles into a heptamer 7, allowing binding of LF and EF, which is then internalized to the cytosol through endocytosis, causing disruption to normal cellular physiology . Antitoxins based on receptor-decoy binding show promising advantages over an antibody-based strategy since it is difficult to engineer toxins to escape the inhibitory effect of the decoy without compromising binding to its cellular receptor. By making the extracellular domain of the main anthrax toxin receptor Capillary Morphogenesis Gene 2 protein recombinantly , that can be used as a prophylaxis or post-exposure treatment, to neutralize anthrax toxins in blood, preventing cell infection. Additionally, fusing an Fc domain to rCMG2 increases the serum half-life through interaction with the salvage neonatal Fc-receptor and lowers renal clearance rate . These factors make rCMG2-Fc a promising anthrax decoy protein, which retains the high binding affinity to the PA along with a longer blood circulatory half-life than rCMG2 .
We used a plant-based expression system for protein expression due to its rapid production rate and inherent scalability, which is critical for providing rapid response under emergency conditions. Moreover, plants rarely carry animal pathogens and are capable of post-translational modification, making them an appealing alternative to traditional protein expression systems such as mammalian cell culture or microbial fermentation . N-glycosylation can affect protein folding, structural integrity, and function , which makes it an important design consideration for glycoprotein based therapeutics. In some cases, proteins with proper glycosylation exhibit optimal efficacy. For example, Fc glycosylation is required to elicit effector functions of human IgG1 . Thus, it should be preserved when immune defense is desired, for instance, when expressing anti tumor mAbs . On the other hand, for drugs that treat chronic conditions, the absence of glycosylation is desired to avoid effector functions and associated inflammatory responses. Another important consideration is that glycosylated proteins are less susceptible to proteases, such as pepsin, compared with aglycosylated counterparts , which should be considered to maximize protein yield. Although the impacts of protein N-glycosylation have been studied, typically only one or two aspects were studied at a time, and these studies were done on antibodies . This study provides a comprehensive approach utilizing a combination of experimental and computational techniques to evaluate the effects of N-glycosylation on rCMG2-Fc fusion protein properties. In this study, the protein expression, toxin neutralization efficacy, binding kinetics, thermostability, and structural configuration were studied experimentally and compared among three rCMG2-Fc glycoform variants.
In addition, we employ atomistic molecular dynamics simulation to understand the structure and dynamics of the predominant glycoform of the APO, ER, and Agly variants. Atomistic MD simulations are well-suited for the study of biomolecular systems, providing full accessibility to virtual, high-resolution, time-ordered, atomic trajectories . MD simulations have been used to study many different biological systems, including lipid membranes, trans-membrane proteins, and other glycoproteins . While fully atomistic protein simulation is a powerful tool to investigate structural and functional information, it is important to recognize the current limitations of the technique. In particular, protein folding is known to occur on the order of microseconds to seconds , while atomistic protein simulation is generally limited to hundreds of nanoseconds due to limited computing resources. This limitation generally prohibits the straightforward simulation of protein fold transitions. The length-scale of atomistic protein simulations is also computationally restricted, allowing only one rCMG2-Fc dimer to be simulated. Despite these limitations, this work shows MD simulation data is capable of providing insight into the effects of glycosylation on protein structure, and improving our understanding and interpretation of experimental observations. To the best of our knowledge, no study has been conducted on Fc-fusion protein considering that manyexperimental and molecular simulation factors. This study provides an integrated experimental and computational approach to evaluate Fc N-glycosylation impacts on rCMG2-Fc properties,4×8 flood tray and potentially serves as a guideline for general glycoprotein based therapeutic design, especially for Fc-fusion proteins.The codon optimized CMG2-Fc sequence includes the extracellular domain of CMG2 , followed by two serine residues, the upper hinge of IgG2 , and Fc region of human IgG1.The resulting sequence corresponds to the APO variant as described previously.A SEKDEL C-terminal motif was included to make the ER variant; a point mutation of N268Q on Fc was included to make the Agly variant. The genes encoding rCMG2-Fc variants were codon-optimized for expression in Nicotiana benthamiana. The full construct consists of the CaMV 35S promoter, Ω leader sequence, gene encoding the Ramy3D signal peptide, followed by rCMG2-Fc gene and octopine synthase terminator . Agrobacterium tumefaciens EHA105 with the helper plasmid was transfected with the resulting binary expression vectors separately via electroporation. A binary vector capable of expressing P19 to suppress RNAi-mediated gene silencing in Nicotiana benthamiana plants was co-infiltrated with the rCMG2-Fc-APO binary vector as previously described .Plant tissue was collected at day 6 after infiltration. To evaluate the average expression level, leaves from 10 plants were collected and stored at −80°C prior to extraction.The leaf powder was weighted and mixed with extraction buffer at a leaf mass to buffer volume ratio of 1:7. The mixture was incubated on a shaker at 4°C for 1 h and then centrifuged at 1,800g at 4°C for 1 h, followed by 0.22 μm filtration to remove insoluble particles.
Filtered plant extract was loaded to protein A column and eluted with glycine-HCl buffer at pH of 3.0. Purified protein was immediately titrated to neutral pH with 1 M tris buffer, and buffer exchanged to 1X PBS through overnight dialysis at 4°C.Expression of rCMG2-Fc in crude plant extract was quantified by a sandwich ELISA. First, ELISA microplate wells were coated with Protein A at a concentration of 50 μg/ml in 1X PBS buffer for 1 h, followed with plate blocking with 5% nonfat milk in 1X PBS buffer for 20 min. Crude plant extracts and purified standards were loaded to the plate and incubated from 1 h . The bound rCMG2-Fc was detected by incubating a horseradish peroxidase -conjugated goat anti human IgG at a concentration of 0.5 μg/ml for 1 h. Plates were washed three times with 1X PBST between each of these steps. All incubation steps were done at 37°C, with an incubation volume of 50 μl. Next, 100 μl of ELISA colorimetric TMB substrate was added to each well and incubated for 10 min, followed by the addition of 100 μl of 1 N HCl to stop the reaction. The absorbance at 450 nm was measured with a microplate reader . The absorbance of protein standard was plotted as a function of rCMG2-Fc concentration, and was fitted to the 4-parameter model in SoftMax Pro software. The concentration of rCMG2-Fc in crude plant extract was determined by interpolating from the linear region of the standard curve.SDS-PAGE and Western blot analyses were performed on purified rCMG2-Fc variants. Protein was denatured and reduced by treating samples at 95°C for 5 min with 5% of 2-mercaptoethanol . For nonreducing SDS-PAGE, samples were denatured by heat treatment at 95°C for 5 min. Samples were loaded to precast 4–20% SDS-Tris HCl polyacrylamide gels , running at 200 V for 35 min. For SDS-PAGE, the gel was washed three times with water and stained with Coomassie Brilliant Blue R-250 Staining Solution . For Western blot analysis, samples were transferred to a nitrocellulose membrane by electrophoretic transfer using the iBlot Gel Transfer Device . For Western blot detecting the CMG2 domain, the membrane was probed with a goat anti-CMG2 polyclonal antibody at a concentration of 0.3 μg/ml, followed by incubation of a polyclonal AP-conjugated rabbit anti-goat IgG antibody at 1:10,000 dilution. For Western blot detecting the Fc domain, the membrane was incubated with a polyclonal AP-conjugated goat anti-human IgG antibody at 1:3,000 dilution. The blots were developed using SIGMAFAST BCIP/NBT according to the product instruction.About 100 ns simulations of Agly, MAN8, and GnGnXF rCMG2-Fc glycoforms were performed in GROMACS with the AMBER ff14SB and GLYCAM06-j force fields. The AMBER topology files were exported to GROMACS format using ACPYPE with updated modifications which enable simulations with the GLYCAM forcefield in GROMACS . The 100 ns production simulations were first preceded by energy minimization in vacuum, solvation, solvated energy minimization, a 100 ps NVT equilibration, and finally a 100 ps NPT equilibration. Both energy minimizations were terminated with a maximum force tolerance of 1,000 kJ mol−1 nm−1. Each glycoform was solvated with explicit water with a minimum distance of 1.2 nm between the glycoprotein and the edge of the periodic box. The solvated systems were then neutralized with either sodium or chloride ions, and then concentrated to 0.155 M NaCl. The velocity-rescale thermostat was used with a reference temperature of 310 K and a time constant of 0.1 ps. The isotropic Parrinello-Rahman barostat was used with a reference pressure of 1 bar, a time constant of 2 ps, and an isothermal compressibility of 4.5 × 10−5 bar−1. All nonbonded interactions employed a short-range cutoff of 1 nm, with vertically shifted potentials such that the potential at the cutoff range is zero. The Particle-Mesh Ewald method with cubic interpolation was used to model long range electrostatic interactions. All non-water bonds were constrained with LINCS , while water bonds were constrained with SETTLE . A 2 fs timestep was used with a sampling interval of 0.1 ns, for a total of 1,000 data points per 100 ns simulation.Recombinant CMG2-Fc variants were transiently expressed in Nicotiana benthamiana whole plants via agroinfiltration under identical conditions, and the expression levels were determined in crude leaf extract at 6 days post infiltration with a sandwich ELISA detecting the Fc domain of rCMG2-Fc. The expression level rankings on the leaf fresh weight basis from high to low were: APO , ER , and Agly variants . Both APO and Agly variants, which only differ in the N-glycosylation generated by a point mutation of N268Q, were targeted to plant Apoplast. The only N-glycosylation site within rCMG2-Fc is located at the CH2 domain of IgG1 Fc . The significantly higher expression of the APO variant with respect to the Agly variant might be due to stabilizing effects of N-glycans on protein accumulation in planta. This observation is consistent with previous studies, where proteins are more susceptible to protease cleavage after deglycosylation . In many cases, targeting proteins to the ER will result in a greater protein yield compared with targeting to the cytosol or apoplast .